Tetrahydrobiopterin
Tetrahydrobiopterin (“BH4”) is a co-factor for the enzymes phenylalanine hydroxylase, tyrosine hydroxylase and tryptophan hydroxylase. The 6R-stereoisomer is believed to be the active form of the BH4 co-factor. The tyrosine hydroxylase and tryptophan hydroxylase enzymes are rate-limiting enzymes in the biosynthesis of neurotransmitters such as dopamine and serotonin. BH4 is considered to be a regulating factor in the biosynthesis of these neurotransmitter amines since BH4 is contained in nerve endings only in an amount which is approximately the Km value of each hydroxylase; a shortage or decrease in enzymes which participate in the biosynthesis of this co-factor from GTP is known to give rise to a decrease in neurotransmitter amines, thus resulting in various neuropsychiatric diseases; researchers (U.S. Pat. No. 5,606,020 to Watanabe et al., which is hereby incorporated by reference) demonstrated that 6R-BH4 enhances the release and liberation of neurotransmitter amines such as dopamine (“DA”), norepinephrine, and serotonin, and that it also enhances the liberation of glutamic acid, aspartic acid or gamma-aminobutyric acid (“GABA”) via DA and the release of acetylcholine via serotonin. On the other hand, it has also been also demonstrated that, when the endogenous BH4 level is lowered by inhibiting the biosynthesis of BH4, the DA level per se is not significantly changed, whereas the release of DA is suppressed by 50-fold or lower. The latter findings suggested that the endogenous BH4 triggers some change in the mechanism of DA release.
Further research (U.S. Pat. No. 5,606,020 to Watanabe et al., which is hereby incorporated by reference) into the molecular mechanism of DA release, isolated, in a membrane fraction of the brain, a receptor site saturated by 6R-BH4 which had both high affinity and high specificity for BH4 and which is the possible regulatory site in the DA release mechanism. It was further found that, because of said specificity, the 6R-BH4 receptor is capable of strictly distinguishing 6R- and 6S-optical isomers of tetrahydrobiopterin and, thus, is capable of selectively recognizing the 6R-isomer, believed to be the active form of the co-factor.
Studies of phenylketonuria have implicated either deficiency in the enzyme phenylalanine hydroxylase (classical PKU) and/or a deficiency in its BH4 co-factor (atypical PKU) as causative agents of this disease (Schaub et al., “Tetrahydrobiopterin Therapy of Atypical Phenylketonuria Due to Defective Dihydrobiopterin Biosynthesis,” Arch. Dis. Child., 53(8):674-676 (1978); Matalon et al., “Screening for Biopterin Defects in Newborns with Phenylketonuria and Other Hyperphenylalaninemias,” Ann. Clin. Lab. Sci., 12(5):411-414 (1982); Kaufman et al., “Use of Tetrahydropterins in the Treatment of Hyperphenylalaninemia Due to Defective Synthesis of Tetrahydrobiopterin: Evidence That Peripherally Administered Tetrahydropterins Enter the Brain,” Pediatrics, 70(3):376-380 (1982); which are hereby incorporated by reference). Autopsied brain specimens and cerebrospinal fluid of patients with Parkinson's disease (Moore et al., “Biopterin in Parkinson's Disease,” J. Neurol. Neurosurg. Psychiatry, 50(1):85-87 (1987); Nagatsu et al., “Biosynthesis of Tetrahydrobiopterin in Parkinsonian Human Brain,” Adv. Neurol., 45:223-226 (1987); which are hereby incorporated by reference), Alzheimer's dementia (Barford et al., “Tetrahydrobiopterin Metabolism in the Temporal Lobe of Patients Dying with Senile Dementia of Alzheimer Type,” J. Neurol. Neurosurg. Psychiatry, 47(7):736-738 (1984), which is hereby incorporated by reference), and infantile autism (Tani et al., “Decrease in 6r-5,6,7,8-tetrahydrobiopterin Content in Cerebrospinal Fluid of Autistic Patients,” Neurosci. Lett., 181 (1-2):169-172 (1994), which is hereby incorporated by reference) show a decrease in BH4 content. Supplementation therapy by BH4 has been studied on a small-scale and proposed for the treatment of phenylketonuria (see references cited above), Parkinson's disease (Curtius et al., “Therapeutic Efficacy of Tetrahydrobiopterin in Parkinson's Disease,” Adv. Neurol., 40:463-466 (1984), which is hereby incorporated by reference), Alzheimer's disease (Aziz et al., “Tetrahydrobiopterin Metabolism in Senile Dementia of Alzheimer Type,” J. Neurol. Neurosurg. Psychiatry, 46(5):410-413 (1983), which is hereby incorporated by reference), depression (Curtius et al., “Successful Treatment of Depression with Tetrahydrobiopterin,” Lancet, 1(8325):657-658 (1983) and infantile autism (Fernell et al., “Possible Effects of Tetrahydrobiopterin Treatment in Six Children with Autism—Clinical and Positron Emission Tomography Data: A Pilot Study,” Dev. Med. Child Neurol., 39(5):313-318 (1997), which is hereby incorporated by reference). Based on these proposals, as of 1997, over a hundred BH4 derivatives had been designed as drugs in Switzerland, the United States and Japan and screened for their co-factor action analogous to BH4. None of these derivatives had demonstrated activity superior to BH4 (U.S. Pat. No. 5,606,020 to Watanabe et al., which is hereby incorporated by reference).
Disappointment with the degree of therapeutic efficacy of BH4 was thought to be due to low penetration of the blood-brain barrier and it was thus suggested that the more lipophilic 6-methyl BH4 (Kaufman et al., “Tetrahydropterin Therapy for Hyperphenylalaninemia Caused by Defective Synthesis of Tetrahydrobiopterin,” Ann. Neurol., 14(3):308-315 (1983), which is hereby incorporated by reference) should improve BH4 therapeutic efficacy by increasing penetration of the blood-brain barrier. Since BH4 is a cofactor needed to generate catecholamine and serotonin, it is possible to find a new use for it, unrelated to PKU or PKU variants.
Attention Deficit Hyperactivity Disorder
Attention deficit hyperactivity disorder (“ADHD”) is a condition characterized by a decreased attention span, hyperactivity, and/or impulsiveness inappropriate for a certain age. Typically, young children have what are known as subtle neurological signs of immaturity. These are involuntary movements of one part of the body that occur while the child is making a voluntary movement of another part of the body. This is referred to as synkinesis, or overflow movements. These overflow movements disappear during normal development and are usually gone by the age of 10. However, in children with ADHD these overflow movements tend to be more intense and last long after the age of 10. This leads researchers to believe there is an abnormality in the maturation and development of the brain areas associated with motor activity in children with ADHD.
Transcranial Magnetic Stimulation (“TMS”) is a non-invasive technique that gives information about brain function and is very useful when studying areas of the nervous system related to motor activity (motor cortex, corticospinal tract, and corpus callosum). A magnetic signal given from a special instrument held close to the patient's head stimulates a small area of the brain that controls a few muscles (for example, the muscles that control one finger). Doctors put electrodes (small pieces of metal taped to areas of the body) over the muscle to measure the electrical activity the muscle produces when it makes a movement. When the magnetic signal activates those muscles, the electrodes pick up and record the electrical activity of the movement that the muscles make in response to the magnetic signal. Researchers are currently studying normal children and those diagnosed with ADHD using TMS to find out if the clinical abnormalities of ADHD are associated with a delay or abnormality in maturation of areas of the nervous system responsible for motor activity (motor cortex and corticospinal tract).
Despite a complete understanding of the cause of ADHD, certain pharmacological agent have been found to be effective in controlling the condition. Specifically, ADHD has been treated with pharmacological agent that mimic catecholamine or serotonin (e.g., psychostimulants, such as methylphenidate (also referred to as RITALIN™)). A Phase III clinical trial is underway to evaluate the benefits and side effects of two medications, clonidine and methylphenidate, used either alone or in combination to treat ADHD in children. Current pharmacotherapeutics for ADHD are psychostimulants, such as RITALIN™; a time-released methylphenidate (CONCERTA™), marketed by McNeil Consumer and Specialty Pharmaceuticals Inc.; ADDERALL™ (an amphetamine), marketed by Richwood Pharmaceutical Co. Inc.; CYLERT™ (pemoline, 2-amino-5-phenyl-2-oxazolin-4-one); DESOXYN™ (methamphetamine), marketed by Abbott Laboratories; and the antihypertensive CATAPRES™ (clonidine, 2-(2,6-dichlorophenylimino)imidazolidine), marketed by Boehringer-Ingelheim.
There is a need for methods and compositions for treating ADHD, and the present invention, in part, is directed to addressing this need.
Hyperphenylalanemia
Hyperphenylalaninemia (“HPA”) is the presence of elevated levels of phenylalanine in the blood, which may result in brain damage and other pathologies. HPA is typically caused by defect(s) in either the BH4 synthetic pathway or the phenylalanine metabolic pathway. The key enzyme in the phenylalanine metabolic pathway is the enzyme phenylalanine-4-hydroxylase (“PAH”). Defects in PAH result in the metabolic disorder Phenylketonuria (“PKU”), which is associated with HPA. Although progress in the neurosciences has been both rapid and broadly based, it has offered little to the practicing pediatrician in regard to treatment of HPA.
Phenylketonuria is a metabolic disease caused by a defect in the activity of PAH, which converts phenylalanine to tyrosine. Accumulation of phenylalanine and its neurotoxic metabolites can produce brain damage in PKU patients, resulting in mental retardation. There are approximately 400 known mutations of PAH resulting in syndromes of varying severity. In approximately 1% of cases, the defect in PAH activity is due to mutation in genes encoding the enzymes such as dihydropteridine reductase (“DHPR”), which are involved in production of the required cofactor (BH4) of PAH.
Although a diet low in phenylalanine can ameliorate the severe retardation associated with untreated PKU, dietary compliance becomes problematic as PKU patients reach adolescence, leading to a rise in phenylalanine blood levels and a loss of intelligence and white matter changes in the brain. Nutritional deficiencies may result from phenylalanine restricted diets, which are typically designed to achieve a safe phenylalanine blood level, which is in the approximate range of 2-6 mg/dL. Furthermore, PKU females of child-bearing age are candidates to have children with Maternal PKU syndrome, characterized by microcephaly, mental retardation and serious congenital heart defects. Investigations into more convenient modes of PKU treatment, other than diet, are needed.
In 1980, Hoskins et al., “Enzymatic Control of Phenylalanine Intake in Phenylketonuria,” Lancet, 1(8165):392-394 (1980), which is hereby incorporated by reference, proposed treatment of PKU via administration of phenylalanine ammonia lyase (“PAL”), a yeast-produced enzyme which converts phenylalanine to cinnamic acid and ammonia, and which, unlike PAH, does not require cofactors. However, limited studies on humans indicated a variable and unpredictable response and the treatment was not cost-effective. Research into PAL therapy was revived in Sarkissian et al., “A Different Approach to Treatment of Phenylketonuria: Phenylalanine Degradation with Recombinant Phenylalanine Ammonia Lyase,” Proc. Natl. Acad. Sci., USA., 96(5):2339-2344 (1999), where it was demonstrated that recombinant PAL lowered blood phenylalanine levels in PKU knockout mice having no PAH activity.
An additional therapeutic regimen for PKU is BH4 loading in patients with residual or low PAH activity or defective BH4 synthesis. Kure et al., “Tetrahydrobiopterin-Responsive Phenylalanine Hydroxylase Deficiency,” J. Pediatr., 135(3):375-378 (1999), which is hereby incorporated by reference, reported a successful response of four PKU patients to oral administration of the BH4 cofactor. Also, Trefz et al., “Successful Treatment of Phenylketonuria with Tetrahydrobiopterin,” Eur. J. Pediatr., 160(5):315 (2001) and Spaapen et al., Tetrahydrobiopterin-Responsive Phenylalanine Hydroxylase Deficiency in Dutch Neonates,” J. Inherit. Metab. Dis., 24(3):352-358 (2001), which are hereby incorporated by reference, have reported similar success with one and five PKU patients in Germany and the Netherlands, respectively. All PKU patients in these studies had normal levels of BH4 prior to treatment, were double heterozygous for PKU mutations with one mutation considered “atypical” (where “atypical”, in this context refers to a phenylalanine level in blood of 6-20 mg/dL)) and responded to BH4 oral administration with normalization of blood levels of phenylalanine, without the need of a low phenylalanine diet.
PKU treatment via administration of the PAH or other enzymes has been limited by protease degradation and rapid clearance of phenylalanine metabolizing enzymes from the gastrointestinal tract. Additionally, oral BH4 loading is limited by the oxidative degradation of BH4 in the digestive tract and the potential need for compliance with a daily multiple dose regimen.
None of the methods for the treatment of HPA, in particular PKU, are entirely satisfactory. Therefore, there is a need for methods and compositions for treating HPA, and the present invention, in part, is directed to addressing this need.